PROJECT SUMMARY/ABSTRACT The proposed experiments are aimed at elucidating the structural and functional properties of a newly discovered family of ion channels, the Otopetrins (Otops). The Otops are just the second class, after Hv1, of proton-selective ion channels described in eukaryotic cells. Otop1 was first discovered in the vestibular system where it is required for the development of calcium carbonate-based otoconia that allow hair cells to detect changes in gravitational forces and acceleration. It was subsequently identified in an unbiased screen for proton channels based on functional analysis of transcripts enriched in taste cells that detect sour. When expressed in Xenopus oocytes and HEK- 293 cells, Otop1 generates a proton-selective ion current that is blocked by extracellular Zn[2+]. The mammalian genome encodes two homologs of Otop1 (Otop2 and Otop3), which also form proton permeable ion channels with distinct functional properties. Here we propose a series of experiments to define mechanisms of gating and permeation of Otop channels, and to identify the underlying structural elements. The CryoEm structure of Otop1 and Otop3 have led us to identify several possible proton permeation pathways; the function of specific residues in permeation or Zn[2+] inhibition will be interrogated using site directed mutagenesis and patch clamp electrophysiology. Other experiments will address the outstanding question of whether Otop channels are gated and identify structural determinants for gating. Members of the otopetrin gene family are widely expressed throughout the body, including in the taste, vestibular and immune systems, in the gastrointestinal tract, and in brown adipose tissue. By understanding the structural and functional properties of each of the Otop isoforms, we will be better able to understand how they contributes to cellular physiology. Moreover, ion channels are the preferred targets for pharmaceuticals and mutations that disrupt their function underlie a growing number of inherited or acquired disorders (channelopathies).